|
| Status |
Public on Jul 13, 2011 |
| Title |
sef1Δ mutant vs Wild type, in YEPD+BPS low iron medium, replicate 5 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Wild type grown in YEPD+BPS (low iron)
|
| Organism |
Candida albicans |
| Characteristics |
strain: SN250
genotype/variation: wild type
|
| Growth protocol |
Saturated overnight cultures of sef1Δ (SN330), hap43Δ (SN694), sfu1Δ (SN515), and isogenic wild type C. albicans (SN250) were inoculated into YPD to OD600=10-4 and incubated with shaking at 30ºC. The next morning, logarithmically growing cells were diluted to OD600 0.01 in iron-replete (wild type and sfu1Δ) or low iron (wild type, sef1Δ, and hap43Δ) medium and incubated at 30°C for 5-6 hours before harvesting at OD600=0.5-0.6. 5-6 biological replicates were performed per strain per condition.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared using a hot phenol method (Miller and Johnson, 2002).
|
| Label |
Cy3
|
| Label protocol |
10 μg of each RNA was treated with DNase I (Turbo DNA-free kit, Ambion) and reverse transcribed using aminoallyl-dUTP and Superscript II (Invitrogen) according to manufacturers’ instructions. cDNA was labeled with Cy3 and Cy5 (Amersham)
|
| |
|
| Channel 2 |
| Source name |
sef1Δ grown in YEPD+BPS (low iron)
|
| Organism |
Candida albicans |
| Characteristics |
strain: SN330
genotype/variation: sef1Δ
|
| Growth protocol |
Saturated overnight cultures of sef1Δ (SN330), hap43Δ (SN694), sfu1Δ (SN515), and isogenic wild type C. albicans (SN250) were inoculated into YPD to OD600=10-4 and incubated with shaking at 30ºC. The next morning, logarithmically growing cells were diluted to OD600 0.01 in iron-replete (wild type and sfu1Δ) or low iron (wild type, sef1Δ, and hap43Δ) medium and incubated at 30°C for 5-6 hours before harvesting at OD600=0.5-0.6. 5-6 biological replicates were performed per strain per condition.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared using a hot phenol method (Miller and Johnson, 2002).
|
| Label |
Cy5
|
| Label protocol |
10 μg of each RNA was treated with DNase I (Turbo DNA-free kit, Ambion) and reverse transcribed using aminoallyl-dUTP and Superscript II (Invitrogen) according to manufacturers’ instructions. cDNA was labeled with Cy3 and Cy5 (Amersham)
|
| |
|
| |
| Hybridization protocol |
Fluorescently labeled cDNAs from mutants were directly hybridized against those from wild type grown under the same conditions; dye flip controls were included. 6 additional arrays were performed using wild type grown under iron-replete vs. iron-limiting conditions. cDNA was labeled with Cy3 and Cy5 (Amersham), and 0.5 μg of each channel was hybridized to custom Agilent C. albicans ORF arrays (15,000 spots/array, 70-mer probes) following a protocol described by Hernday et al. (Methods Enzymol. 2010;470:737-58)
|
| Scan protocol |
Arrays were scanned using a Genepix 4000A Axon scanner, and spots were filtered using GenePix Pro software.
|
| Description |
Biological replicate 5 of 6. Low iron, Wt vs sef1DD
|
| Data processing |
Data were normalized using Goulphar (LOWESS normalization) and subjected to One-class Significance Analysis of Microarrays (SAM) analysis with a median false discovery rate of 0.1%. Candidates meeting SAM criteria were also required to exhibit median 2-fold changes among 5-6 experiments.
|
| |
|
| Submission date |
Jul 12, 2011 |
| Last update date |
Jul 13, 2011 |
| Contact name |
Changbin Chen |
| E-mail(s) |
[email protected]
|
| Phone |
4154768992
|
| Fax |
4154768201
|
| Organization name |
University of California, San Francisco
|
| Department |
Department of Microbiology & Immunology
|
| Lab |
Dr. Suzanne Noble LAB
|
| Street address |
HSE450, 513 Parnassus Avenue
|
| City |
San Francisco |
| State/province |
California |
| ZIP/Postal code |
94143 |
| Country |
USA |
| |
|
| Platform ID |
GPL13813 |
| Series (2) |
| GSE30590 |
A unique iron homeostasis regulatory circuit with reciprocal roles in Candida albicans commensalism and pathogenesis [Expression Array] |
| GSE30593 |
A unique iron homeostasis regulatory circuit with reciprocal roles in Candida albicans commensalism and pathogenesis |
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